- Email: [email protected]
High throughput genotyping of apoE using molecular beacons and real-time monitoring of fluorescence PCR
Wednesday June 28, 2000: Poster Abstracts P:H5 Genetic Screening
216
4- 12 vs. 60 4- 14 cpm x 104/3-cm dish; n = 6, P < 0.05). After pretreatment of oxLDL with 200 #g/mL recombinant PAF acetylhydrolase for 1 hour, apoptosis, bFGF down-regulation, and inhibition of DNA synthesis were negligible. The PAF receptor antagonist WEB 2086 (10 /,M) significantly suppressed oxLDL effects. However, lyopnilized PAF (up to 1/zM) was only weakly active compared to oxLDL. Effects of oxLDL were almost entirely prevented by supplementation of exogenous bFGF (10 ng/mL). Condnsions: In endothelial cells, oxLDL promotes apoptosis, inhibits DNA synthesis, and down-regulates bFGF without membrane disruption. These effects are mediated by phospholipids that act through PAF receptors as indicated by their susceptibility to sN2 acetylhydrolysis and inhibition by the PAF antagonist. Effects of bFGF suggest bFGF's pivotal role in endothelial response to oxLDL, a key process of atherogenesis.
WeP42:W26 ] Mast cell-mediated downregulation of Bcl-2 expression triggers endothelial cell apoptosis in vitro S. U,itti, N. Shiota, M. Leskinen, Y. Wang, P. Kovanen, K. Lindstedt. Wihuri
Research Institute, Helsinki, Finland
Objectives: Mast cells are present in coronary atheromas where they localize to the shoulder region, the site of erosion or rupture in myocardial infarction. These mast cells are known to express TNF-ot. a proinflammatory cytokine capable of inducing endothelial cell dysfunction and apoptosis. Since the presence of an intact, functional endothelium is critical for the stability of the atherosclerotic plaque, mast cells might participate in its destablization and rupture by affecting the integrity of the endothelium. Methods and Results: Here we show, by means of RT-PCR, western blotting, immunocytochemistry, propidium iodide-staining and DNA-laddering, that in isolated and cultured rat myocardial endothelial cells, stimulation of isolated rat serosal mast cells with ensuing degranulation did downregulate the expression of the anti-apoptotic protein Bcl-2, with subsequent induction of apoptosis. The mast cell-mediated apoptotic effect, which resided in the granule remnant-fraction, was comparable to the effect obtained with purified TNF-ot. No effect was seen with another known inducer of apoptosis, i.e chymase, also present in the mast cell granule remnants. The Bcl-2 mRNA expression level was downregulated rapidly (30 min), and was followed by a major decrease in Bcl-2 protein. In contrast, there was no significant change in the expression level of either Bax, a pro-apototic protein, or p53. As a consequence, cytochrome c was found to be increased and rapidly released from mitochondria into cytoplasm. Conclusions: Mast cell stimulation and degranulation results in a rapid downregulation of Bcl-2 mRNA- and protein-levels followed by the induction of endothelial cell apoptosis. Thus, mast cells may participate in the erosion of atherosclerotic plaques by inducing endothelial cell apoptosis.
P:H5
GENETIC SCREENING
W e P I : H 5 ] Effects ofpolymorphism of angiotensin-converting enzyme,
apolipoprotein E and endothelial nitric oxide synthase genes on progression of arterial wall thickening in Japanese general population M. Komatsu-Matsuyoshi1, T. Kawagishi 1, M. Emoto 1, T. Shoji I , H. Kanda I , A. Yamada 1, M. Fukumoto 1, H. Taniwaki 1, M. Hosoi 2, Y. Okuno 1, Y. Nishizawa 1. 1Second Department of lnternal Medcine, Osaka City
University; 21nternal Medcine, Osaka Oty General Hospital, Osaka, Japan
eNOS gene did not affect the progression of arterial wall thickening during 3 years follow-up study periods. Results: ACE gene polymorphism is associated with the progression of the carotid arterial wall thickening. Conclusions: The D allele of the ACE gene may be a genetic determinant of the carotid arterial wall thickening in Japanese general population.
WeP2:H5 ] Polymorphisms in apo E gene promoter. Frequency in Spanish newborns, elderly and coronary patients. Relationship with coronary stenosis levels A. Gafi~n 1, A.L. Garcia-Offn I , J.M. Casado 2, C. Gonzalvo 1, J. Puzo 3, A. Cenarro4, M. Pocovf 1, F. Civeira4 ./Departamento de Bioqu(mica y
Biologia Molecular y Celular, Universidad de Zaragoza; 2Servicio de Cardiolog(a; 4Laboratorio de Investigacidn Molecular y Medicina lnterna, Hospital Miguel Servet, Zaragoza; 3Hospital San Jorge, Huesca, Spain Apolipoprotein E (apt E) macrophage expression in vascular tissue is related with colesterol efflux and the atherosclerotic lesions development. It has been demonstrated that two polymorphims in the apt E gene promoter, - 4 9 1 A / T and - 2 1 9 G/T modify the expression in hepatocytes and astrocytes. Objective: To determine the frequency of - 4 9 1 A/I" and - 2 1 9 G,rr polymorphisms in Spanish newborns, elderly and coronary patients and its possible association with the coronary lesion extent. Methods: Both polymorpnisms and a p t E genotype were determined by PCR and restriction analysis in 103 newborns (NB), 115 non coronary population > 70 years old (EL) and 147 men < 60 years old with coronary lesions quantified by angiographic study (CP). Results: The frequencies of - 4 9 1 A and - 2 1 9 T were 0.82 and 0.45 in NB, 0.81 and 0.43 in EL and 0.82 and 0.41 in CP, respectively. In the CP group, - 2 1 9 T/T patients have nigher coronary extent scores than - 2 1 9 GIG patients, although these values did not reach statistical significance. Conclusions: Our data indicate that there are no significant diferences in allelic frequencies between NB, EL, CP groups but a relationship could exist between - 2 1 9 T/G polymorphism and the extent of coronary lesions.
I WeP3:H5 I Spectrum of mutations in LDL receptor gene in Czech )
hypercbolesterolemic patients
V. Kuhmv~iI , H. Francov~i I , V. So~ka2, L. Fajkusov~i I , T. Freiberger I .
1Research Institute of ChiM Health; 2 University Hospital, Brno, Czech Republic Objective: The purpose of our study was to identify molecular determinants for the development of hyperlipidemia and/or athero sclerosis in Czech population. Especially for classical FH, when an impairment of the LDL receptor function due to mutations is one of the most common metabolic disorder. In our country there is no evidence of mutation spectrum in LDL receptor gene still. Methods: Patients with high total and LDL cholesterol, normal serum triglyceride and with family history of premature CHD were selected for the study. DNA sequence variations were detected using heteroduplex, SSCP, DGGE, PCR/RFLP analysis and in detail characterized by DNA sequencing. Results: Molecular searching for mutations in the coding and flanking intronic sequences of the low density lipoprotein receptor gene, resulted in identification of 18 sequence variations, 13 of which are new and were not described previously. Conclusions: Knowledge of mutations causing classical FH is an aid for unambiguous diagnosis, facilitates genetic consulting at early age together with preventing disease manifestation. This study was supported by grant IGA MH CZ, NE/5554-3.
Objective: To investigate the effects of the polymorphism of angiotensinconverting enzyme (ACE), apolipoprotein E (apt E) and endothelial nitric oxide synthase (eNOS) genes on the progression of arterial wall thickening in Japanese general population. Methods: A nigh-resolution B-mode ultrasonograpnic examinations of the common carotid and femoral arterial intima-medial thickness (IMT) were repeated after a follow-up of 36 months for 39 men and 99 women who were recruited from participants of a local health chech program. The IMT increased by 0.05 4- 0.004 mm in the carotid artery and by 0.07 4- 0.006 m m in the femoral artery. The increase in carotid IMT was significantly higher in the subjects with D allele of the ACE gene than in those without (p < 0.0005). Multiple regression analysis showed that cigarette-years and presence of the D allele of the ACE gene were the strongest predictors of the progression of carotid IMT (R 2 = 0.247, p < 0.001). The polymorpnism of the apo E or
WeP4:H5 ] High throughput genotyping of apoE using molecular
beacons and real-time monitoring of fluorescence PCR P. Hanifi Moghaddam [ , K. Szuhai 2, A. Tholens I , A.K. Raap 2, R.R. Frants 1.
Departments of l Human and Clinical Genetics; 2Molecular Cell-Biology, Leiden University Medical Center, Leiden, The Netherlands The Apolipoprotein E gene (APOE), a key player of the lipid transport, exist as three main alleles in the general population: E2, E3 and EA. These polymorpnisms correspond to mutations in the coding sequence of the APOE gene resulting in amino acid substitution (Arg and Cys) at position 112 and 158 of the protein: E3 (Cys 112, Arg 158), E4 (Arg 112, Arg 158) and E2 (Cys112, Cys 158). This allelic variation contributes to susceptibility to atherosclerotic cardiovascular disease and Alzheimer disease.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
Wednesday June 28, 2000: Poster Abstracts P:H5 Genetic Screening To date, genotyping for APOE is mainly performed with RFLP analysis, and separation of the DNA fragments on acrylamide gels. This method is very consuming and gives rise to unequivocal results even after optimisation of PCR and restriction reactions. The aim of this study was to establish and apply a novel methodology, based on Molecular Beacons, for high-throughput sequence variant analysis of the APOE gene. Molecular Beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogenous solutions with excellent allele discrimination. When they bind to their targets they undergo a conformational change that restores the fluorescence of an internally quenched fluorophore. We designed Molecular Beacons to detect DNA variations at the positions 112 and 158 of the APOE genes. This method correctly identified the three major isoforms of the APOE: E2, E3 and FA. The assay was carried out entirely in sealed PCR tubes and was simple to perform and interpret. Molecular Beacons PCR enabled the high throughput, rapid (96 samples in 2 hours), easy to perform and reliable genotyping of APOE alleles.
,IWeP5:H5 ] CETP mutations identified in Caucasians with high or low HDL cholesterol I
B. Agerholm-Larsen, B.G. Nordestgaard, P. Schnohr, R. Steffensen, A. Tybjaerg-Hansen. University of Copenhagen, Denmark
Objective: To identify genetic variation in cholesteryl ester transfer protein (CETP) gene influencing HDL cholesterol levels; CETP mediate transfer of cholesteryl ester from high-density lipoprotein (HDL) in exchange for triglycerides in apolipoprotein B containing lipoproteins. Methods: We selected 200 subjects among 9300 women and men from the Copenhagen City Heart Study with HDL cholesterol > 99 th or < 1st percentile. We sequenced regions thought to involve lipid binding in the CETP gene, exons 12 through 16, in these 200 subjects using 4 different PCR setups. Results: We found the relative frequency of 373P carriers (24%), and 454Q carders (21%) to be higher among subjects with low HDL cholesterol compared with subjects with high HDL cholesterol (5.5% and 3.7%, respectively, P < 0.001). We also found the relative frequency of 450V careers was 55% in subjects with high HDL cholesterol and 44% in subjects with low HDL cholesterol (P = 0.18). Among subjects with high HDL cholesterol 26% was carriers of a 12 bp insertion in intron 12 compared with 18% among subjects with low HDL cholesterol (P = 0.20). An G for A substitution 14 bp upstream from the intronl2/exonl3 splice site had a similar frequency in both groups of subjects (P = 0.72). We also found one subject with a T for C substitution at codon 370.
217
Conclusions: The 373P and the 451Q mutations in CETP are overrepresented in Caucasians with extreme low levels of HDL cholesterol, while the 405V mutation was only slightly overrepresented in subjects with extremely high levels of HDL cholesterol.
I WeP6:H5
[ Identification of differentially expressed genes in macrophages from rabbits with high (lIAR) and low (LAR) atherosclerotic response
R. Burkhardt, D. Teupser, J. Thiery. Institute of Clinical Chemistry, University Hospital LMU, Munich, Germany
Objective: We have previously described two strains of New Zealand White rabbits with high (HAR) and low (LAR) atherosclerotic response to diet induced hypercholesterolemia. The aim of the present study was to identify new candidate genes of atherosclerosis susceptibility and resistance in macrophages from both strains of rabbits. Methods: mRNA was isolated from peritoneal macrophages of 6 HAR and 6 LAR rabbits fed a 0.5% cholesterol diet for 21 days. Using a RT-PCR based suppression subtractive hybridization assay we either enriched for genes with higher expression in LAR rabbits or with higher expression in HAR rabbits. The enriched cDNAs were inserted into a T/A-cloning vector. We then rescreened for clones representing mRNAs that were truly differentially expressed and determined their sequences. The sequences were compared with a nucleotide sequence database (GenBank). Results: Several of the sequences identified in macrophages from both strains of rabbits showed no homology with genes in the database. However, at present we have identified two cDNAs with higher expression in macrophages from HAR rabbits, which show homology with human NUMB protein mRNA (89%) and the human mRNA for the KIAA 0196 gene (93%). Among the known genes with higher expression in macrophages from LAR rabbits we found a high homology with human arginase (91%), rabbit fibronectin mRNA (99%) and rabbit ferritin H-chain mRNA (98%). Conclusion: Suppression subtractive hybridization is suitable to identify differentially expressed genes in hypercholesterolemic rabbits with high and low atherosclerotic response. Some of the genes identified so far, are involved in macrophage differentiation and uptake mechanisms. Ongoing studies will provide evidence about the role of the identified genes in atherogenesis or atherosclerosis prevention.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
216
4- 12 vs. 60 4- 14 cpm x 104/3-cm dish; n = 6, P < 0.05). After pretreatment of oxLDL with 200 #g/mL recombinant PAF acetylhydrolase for 1 hour, apoptosis, bFGF down-regulation, and inhibition of DNA synthesis were negligible. The PAF receptor antagonist WEB 2086 (10 /,M) significantly suppressed oxLDL effects. However, lyopnilized PAF (up to 1/zM) was only weakly active compared to oxLDL. Effects of oxLDL were almost entirely prevented by supplementation of exogenous bFGF (10 ng/mL). Condnsions: In endothelial cells, oxLDL promotes apoptosis, inhibits DNA synthesis, and down-regulates bFGF without membrane disruption. These effects are mediated by phospholipids that act through PAF receptors as indicated by their susceptibility to sN2 acetylhydrolysis and inhibition by the PAF antagonist. Effects of bFGF suggest bFGF's pivotal role in endothelial response to oxLDL, a key process of atherogenesis.
WeP42:W26 ] Mast cell-mediated downregulation of Bcl-2 expression triggers endothelial cell apoptosis in vitro S. U,itti, N. Shiota, M. Leskinen, Y. Wang, P. Kovanen, K. Lindstedt. Wihuri
Research Institute, Helsinki, Finland
Objectives: Mast cells are present in coronary atheromas where they localize to the shoulder region, the site of erosion or rupture in myocardial infarction. These mast cells are known to express TNF-ot. a proinflammatory cytokine capable of inducing endothelial cell dysfunction and apoptosis. Since the presence of an intact, functional endothelium is critical for the stability of the atherosclerotic plaque, mast cells might participate in its destablization and rupture by affecting the integrity of the endothelium. Methods and Results: Here we show, by means of RT-PCR, western blotting, immunocytochemistry, propidium iodide-staining and DNA-laddering, that in isolated and cultured rat myocardial endothelial cells, stimulation of isolated rat serosal mast cells with ensuing degranulation did downregulate the expression of the anti-apoptotic protein Bcl-2, with subsequent induction of apoptosis. The mast cell-mediated apoptotic effect, which resided in the granule remnant-fraction, was comparable to the effect obtained with purified TNF-ot. No effect was seen with another known inducer of apoptosis, i.e chymase, also present in the mast cell granule remnants. The Bcl-2 mRNA expression level was downregulated rapidly (30 min), and was followed by a major decrease in Bcl-2 protein. In contrast, there was no significant change in the expression level of either Bax, a pro-apototic protein, or p53. As a consequence, cytochrome c was found to be increased and rapidly released from mitochondria into cytoplasm. Conclusions: Mast cell stimulation and degranulation results in a rapid downregulation of Bcl-2 mRNA- and protein-levels followed by the induction of endothelial cell apoptosis. Thus, mast cells may participate in the erosion of atherosclerotic plaques by inducing endothelial cell apoptosis.
P:H5
GENETIC SCREENING
W e P I : H 5 ] Effects ofpolymorphism of angiotensin-converting enzyme,
apolipoprotein E and endothelial nitric oxide synthase genes on progression of arterial wall thickening in Japanese general population M. Komatsu-Matsuyoshi1, T. Kawagishi 1, M. Emoto 1, T. Shoji I , H. Kanda I , A. Yamada 1, M. Fukumoto 1, H. Taniwaki 1, M. Hosoi 2, Y. Okuno 1, Y. Nishizawa 1. 1Second Department of lnternal Medcine, Osaka City
University; 21nternal Medcine, Osaka Oty General Hospital, Osaka, Japan
eNOS gene did not affect the progression of arterial wall thickening during 3 years follow-up study periods. Results: ACE gene polymorphism is associated with the progression of the carotid arterial wall thickening. Conclusions: The D allele of the ACE gene may be a genetic determinant of the carotid arterial wall thickening in Japanese general population.
WeP2:H5 ] Polymorphisms in apo E gene promoter. Frequency in Spanish newborns, elderly and coronary patients. Relationship with coronary stenosis levels A. Gafi~n 1, A.L. Garcia-Offn I , J.M. Casado 2, C. Gonzalvo 1, J. Puzo 3, A. Cenarro4, M. Pocovf 1, F. Civeira4 ./Departamento de Bioqu(mica y
Biologia Molecular y Celular, Universidad de Zaragoza; 2Servicio de Cardiolog(a; 4Laboratorio de Investigacidn Molecular y Medicina lnterna, Hospital Miguel Servet, Zaragoza; 3Hospital San Jorge, Huesca, Spain Apolipoprotein E (apt E) macrophage expression in vascular tissue is related with colesterol efflux and the atherosclerotic lesions development. It has been demonstrated that two polymorphims in the apt E gene promoter, - 4 9 1 A / T and - 2 1 9 G/T modify the expression in hepatocytes and astrocytes. Objective: To determine the frequency of - 4 9 1 A/I" and - 2 1 9 G,rr polymorphisms in Spanish newborns, elderly and coronary patients and its possible association with the coronary lesion extent. Methods: Both polymorpnisms and a p t E genotype were determined by PCR and restriction analysis in 103 newborns (NB), 115 non coronary population > 70 years old (EL) and 147 men < 60 years old with coronary lesions quantified by angiographic study (CP). Results: The frequencies of - 4 9 1 A and - 2 1 9 T were 0.82 and 0.45 in NB, 0.81 and 0.43 in EL and 0.82 and 0.41 in CP, respectively. In the CP group, - 2 1 9 T/T patients have nigher coronary extent scores than - 2 1 9 GIG patients, although these values did not reach statistical significance. Conclusions: Our data indicate that there are no significant diferences in allelic frequencies between NB, EL, CP groups but a relationship could exist between - 2 1 9 T/G polymorphism and the extent of coronary lesions.
I WeP3:H5 I Spectrum of mutations in LDL receptor gene in Czech )
hypercbolesterolemic patients
V. Kuhmv~iI , H. Francov~i I , V. So~ka2, L. Fajkusov~i I , T. Freiberger I .
1Research Institute of ChiM Health; 2 University Hospital, Brno, Czech Republic Objective: The purpose of our study was to identify molecular determinants for the development of hyperlipidemia and/or athero sclerosis in Czech population. Especially for classical FH, when an impairment of the LDL receptor function due to mutations is one of the most common metabolic disorder. In our country there is no evidence of mutation spectrum in LDL receptor gene still. Methods: Patients with high total and LDL cholesterol, normal serum triglyceride and with family history of premature CHD were selected for the study. DNA sequence variations were detected using heteroduplex, SSCP, DGGE, PCR/RFLP analysis and in detail characterized by DNA sequencing. Results: Molecular searching for mutations in the coding and flanking intronic sequences of the low density lipoprotein receptor gene, resulted in identification of 18 sequence variations, 13 of which are new and were not described previously. Conclusions: Knowledge of mutations causing classical FH is an aid for unambiguous diagnosis, facilitates genetic consulting at early age together with preventing disease manifestation. This study was supported by grant IGA MH CZ, NE/5554-3.
Objective: To investigate the effects of the polymorphism of angiotensinconverting enzyme (ACE), apolipoprotein E (apt E) and endothelial nitric oxide synthase (eNOS) genes on the progression of arterial wall thickening in Japanese general population. Methods: A nigh-resolution B-mode ultrasonograpnic examinations of the common carotid and femoral arterial intima-medial thickness (IMT) were repeated after a follow-up of 36 months for 39 men and 99 women who were recruited from participants of a local health chech program. The IMT increased by 0.05 4- 0.004 mm in the carotid artery and by 0.07 4- 0.006 m m in the femoral artery. The increase in carotid IMT was significantly higher in the subjects with D allele of the ACE gene than in those without (p < 0.0005). Multiple regression analysis showed that cigarette-years and presence of the D allele of the ACE gene were the strongest predictors of the progression of carotid IMT (R 2 = 0.247, p < 0.001). The polymorpnism of the apo E or
WeP4:H5 ] High throughput genotyping of apoE using molecular
beacons and real-time monitoring of fluorescence PCR P. Hanifi Moghaddam [ , K. Szuhai 2, A. Tholens I , A.K. Raap 2, R.R. Frants 1.
Departments of l Human and Clinical Genetics; 2Molecular Cell-Biology, Leiden University Medical Center, Leiden, The Netherlands The Apolipoprotein E gene (APOE), a key player of the lipid transport, exist as three main alleles in the general population: E2, E3 and EA. These polymorpnisms correspond to mutations in the coding sequence of the APOE gene resulting in amino acid substitution (Arg and Cys) at position 112 and 158 of the protein: E3 (Cys 112, Arg 158), E4 (Arg 112, Arg 158) and E2 (Cys112, Cys 158). This allelic variation contributes to susceptibility to atherosclerotic cardiovascular disease and Alzheimer disease.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
Wednesday June 28, 2000: Poster Abstracts P:H5 Genetic Screening To date, genotyping for APOE is mainly performed with RFLP analysis, and separation of the DNA fragments on acrylamide gels. This method is very consuming and gives rise to unequivocal results even after optimisation of PCR and restriction reactions. The aim of this study was to establish and apply a novel methodology, based on Molecular Beacons, for high-throughput sequence variant analysis of the APOE gene. Molecular Beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogenous solutions with excellent allele discrimination. When they bind to their targets they undergo a conformational change that restores the fluorescence of an internally quenched fluorophore. We designed Molecular Beacons to detect DNA variations at the positions 112 and 158 of the APOE genes. This method correctly identified the three major isoforms of the APOE: E2, E3 and FA. The assay was carried out entirely in sealed PCR tubes and was simple to perform and interpret. Molecular Beacons PCR enabled the high throughput, rapid (96 samples in 2 hours), easy to perform and reliable genotyping of APOE alleles.
,IWeP5:H5 ] CETP mutations identified in Caucasians with high or low HDL cholesterol I
B. Agerholm-Larsen, B.G. Nordestgaard, P. Schnohr, R. Steffensen, A. Tybjaerg-Hansen. University of Copenhagen, Denmark
Objective: To identify genetic variation in cholesteryl ester transfer protein (CETP) gene influencing HDL cholesterol levels; CETP mediate transfer of cholesteryl ester from high-density lipoprotein (HDL) in exchange for triglycerides in apolipoprotein B containing lipoproteins. Methods: We selected 200 subjects among 9300 women and men from the Copenhagen City Heart Study with HDL cholesterol > 99 th or < 1st percentile. We sequenced regions thought to involve lipid binding in the CETP gene, exons 12 through 16, in these 200 subjects using 4 different PCR setups. Results: We found the relative frequency of 373P carriers (24%), and 454Q carders (21%) to be higher among subjects with low HDL cholesterol compared with subjects with high HDL cholesterol (5.5% and 3.7%, respectively, P < 0.001). We also found the relative frequency of 450V careers was 55% in subjects with high HDL cholesterol and 44% in subjects with low HDL cholesterol (P = 0.18). Among subjects with high HDL cholesterol 26% was carriers of a 12 bp insertion in intron 12 compared with 18% among subjects with low HDL cholesterol (P = 0.20). An G for A substitution 14 bp upstream from the intronl2/exonl3 splice site had a similar frequency in both groups of subjects (P = 0.72). We also found one subject with a T for C substitution at codon 370.
217
Conclusions: The 373P and the 451Q mutations in CETP are overrepresented in Caucasians with extreme low levels of HDL cholesterol, while the 405V mutation was only slightly overrepresented in subjects with extremely high levels of HDL cholesterol.
I WeP6:H5
[ Identification of differentially expressed genes in macrophages from rabbits with high (lIAR) and low (LAR) atherosclerotic response
R. Burkhardt, D. Teupser, J. Thiery. Institute of Clinical Chemistry, University Hospital LMU, Munich, Germany
Objective: We have previously described two strains of New Zealand White rabbits with high (HAR) and low (LAR) atherosclerotic response to diet induced hypercholesterolemia. The aim of the present study was to identify new candidate genes of atherosclerosis susceptibility and resistance in macrophages from both strains of rabbits. Methods: mRNA was isolated from peritoneal macrophages of 6 HAR and 6 LAR rabbits fed a 0.5% cholesterol diet for 21 days. Using a RT-PCR based suppression subtractive hybridization assay we either enriched for genes with higher expression in LAR rabbits or with higher expression in HAR rabbits. The enriched cDNAs were inserted into a T/A-cloning vector. We then rescreened for clones representing mRNAs that were truly differentially expressed and determined their sequences. The sequences were compared with a nucleotide sequence database (GenBank). Results: Several of the sequences identified in macrophages from both strains of rabbits showed no homology with genes in the database. However, at present we have identified two cDNAs with higher expression in macrophages from HAR rabbits, which show homology with human NUMB protein mRNA (89%) and the human mRNA for the KIAA 0196 gene (93%). Among the known genes with higher expression in macrophages from LAR rabbits we found a high homology with human arginase (91%), rabbit fibronectin mRNA (99%) and rabbit ferritin H-chain mRNA (98%). Conclusion: Suppression subtractive hybridization is suitable to identify differentially expressed genes in hypercholesterolemic rabbits with high and low atherosclerotic response. Some of the genes identified so far, are involved in macrophage differentiation and uptake mechanisms. Ongoing studies will provide evidence about the role of the identified genes in atherogenesis or atherosclerosis prevention.
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000